Cells Signal Transduction Cascades in Endothelial Complement C3a

Complement C3a and C5a Induce Different Signal Transduction Cascades in Endothelial Cells

This information is current as Ingrid U. Schraufstatter, Khanh Trieu, Lyudmila Sikora, P. of September 26, 2021. Sriramarao and Richard DiScipio J Immunol 2002; 169:2102-2110; ; doi: 10.4049/jimmunol.169.4.2102 http://www.jimmunol.org/content/169/4/2102 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Complement C3a and C5a Induce Different Signal Transduction Cascades in Endothelial Cells1

Ingrid U. Schraufstatter,2* Khanh Trieu,* Lyudmila Sikora,† P. Sriramarao,† and Richard DiScipio†

In leukocytes, C3a and C5a cause chemotaxis in a Gi-dependent, pertussis toxin (PT)-sensitive fashion. Because we found that HUVECs and immortalized human dermal microvascular endothelial cells express small numbers of C3aRs and C5aRs, we asked what the function of these receptors was on these cells. Activation of the C3aR caused transient formation of actin stress ﬁbers, which was not PT-sensitive, but depended on rho activation implying coupling to G␣12 or G␣13. Activation of the C5aR caused a delayed and sustained cytoskeletal response, which was blocked by PT, and resulted in cell retraction, increased paracellular permeability, and facilitated eosinophil transmigration. C5a, but not C3a, was chemotactic for human immortalized dermal microvascular endothelial cells. The response to C5a was blocked by inhibitors of phosphatidylinositol-3-kinase, src kinase, and Downloaded from of the epidermal growth factor (EGF) receptor (EGFR) as well as by neutralizing Abs against the EGFR and heparin-binding EGF-like factor. Furthermore, immune precipitations showed that the EGFR was phosphorylated following stimulation with C5a. The C5aR in endothelial cells thus uses a signaling cascade–transactivation of the EGFR–that does not exist in leukocytes, while the C3aR couples to a different G protein, presumably G␣12/13. The Journal of Immunology, 2002, 169: 2102–2110.

omplement activation leads to the production of the ana- tion of these receptors on cell types other than leukocytes is http://www.jimmunol.org/ phylatoxins C3a and C5a, which are basic polypeptides under active investigation. C of 74–77 aa derived from the ␣-chains of their parent Both the C3aR and the C5aR are GPCRs, which couple to the proteins. C3a and C5a are known for their stimulatory effect on pertussis toxin (PT)-sensitive Gi␣ (21–24) and to the PT-insensi- leukocytes including chemotaxis of neutrophils, eosinophils, and tive G␣16 (25, 26) in leukocytes. Leukocytes are very rich in Gi␣, monocytes (1–5). Furthermore, the anaphylatoxins can also induce and G␣16 expression is limited to cells of hemopoietic lineage (27). smooth muscle contraction (6) and can evoke an increase in vas- Because G protein usage is not absolute, it is conceivable that other cular permeability (7, 8). cell types use additional G proteins and/or signal transduction However, it has been appreciated only recently that the expres- cascades. by guest on September 26, 2021 sion of anaphylatoxin receptors is not limited to leukocytes. In In this study, we report that cultured human endothelial cells particular, expression of the C5aR is widespread and has been express C3aRs and C5aRs, and that the two receptors use different shown in liver, lung, kidney, and the CNS (9–12). Although it was signal transduction cascades to activate cellular responses distinct reported that expression of C5aRs is low on endothelial cells (13, from those on leukocytes. 14), it is incorrect to conclude that activation of these receptors is functionally irrelevant, because engagement of a few hundred G Materials and Methods protein-coupled receptors (GPCRs)3 per cell is sufficient for Purification of C3a and C5a maximal cell activation (15). Indeed, it has been suggested that Human C3a and C5a were produced after cleavage of their parent proteins, endothelial cell-derived superoxide generation played a role in C3 and C5, respectively, using a fluid phase C3/C5 convertase, CVF, and C5a-mediated pulmonary injury (16). Still less is known about Bb (28). C3a and C5a were collected after direct passage through DEAE- C3aRs on nonhemopoietic cells. Their expression has been de- Sephacel equilibrated in 20 mM imidazole HCl (pH 7)/0.075 M NaCl. Then, the anaphylatoxins were adsorbed onto CM-Sephadex (Amersham scribed in the CNS (17, 18), on epithelial cells (19), and on Pharmacia Biotech, Piscataway, NJ), and eluted with 0.25 M NaCl. The endothelial cells following ischemia/reperfusion (20). The func- synthetic polypeptide consisting of the 21 terminal amino acids of C3a, C3a (57–77), was a kind gift of Dr. T. E. Hugli (La Jolla Institute for Molecular Medicine, San Diego, CA). Cell culture Departments of *Cancer Biology and †Vascular Biology, La Jolla Institute for Mo- lecular Medicine, San Diego, CA 92121 HUVECs were obtained from Clonetics (San Diego, CA) and used between passage 5 and 7. Human immortalized dermal microvascular endothelial Received for publication December 19, 2001. Accepted for publication June 19, 2002. cells (HMECs; Ref. 29) were obtained from the Center for Disease Control The costs of publication of this article were defrayed in part by the payment of page (Atlanta, GA). The cells were grown in endothelial growth medium as charges. This article must therefore be hereby marked advertisement in accordance recommended by the supplier (Clonetics). with 18 U.S.C. Section 1734 solely to indicate this fact. HMECs or HUVECs were seeded at low density on collagen-coated 1 This work was supported by National Institute Health Grants HL55657 (to I.U.S.) coverslips and grown in endothelial growth medium (Clonetics) containing and AI35796 (to P.S.). 10% FCS. On the day of the experiment, cells were serum starved for 2 h 2 Address correspondence and reprint requests to Dr. Ingrid U. Schraufstatter, De- (HUVECs) or 3–4 h (HMECs) and stimulated with C3a or C5a for the time partment of Cancer Biology, La Jolla Institute for Molecular Medicine, 4570 Exec- indicated for each experiment. For immuneprecipitations, HMECs were utive Drive, #100, San Diego, CA 92121. E-mail address: [email protected] serum starved for 16 h. All experiments were performed at 37°C in a tissue 3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; HMEC, hu- culture incubator. man immortalized dermal microvascular endothelial cell; EGF, epidermal growth To block Gi␣, endothelial cells were incubated for 16 h with 100 ng/ml factor; EGFR, EGF receptor; PT, pertussis toxin; LPA, lysophosphatidic acid; HB- of PT (List Biological Laboratories, Campbell, CA). Rho was inhibited EGF, heparin-binding EGF-like factor; F-actin, filamentous actin. with C3 botulinum toxin (10 ␮g/ml; List Biological Laboratories) for 24 h,

which leads to inactivation of rho in ϳ90% of endothelial cells (30). Rho Immuneprecipitations of the EGFR ␮ kinase, a downstream target of rho, was inhibited with 10 M Y27632 (gift 2 of Yoshitomi Pharmaceuticals, Osaka, Japan) added 30 min before the HMECs were grown to confluence on 100-mm tissue culture plates, serum addition of the stimulus (31). Similarly, tyrphostin AG 1478 (1 ␮Min starved for 18 h, incubated with 5 ml fresh serum-free media, stimulated DMSO; Calbiochem, La Jolla, CA), a specific inhibitor of the epithelial with 50 nM C5a, 500 nM C3a, or 30 ng/ml of EGF for the indicated times ␮ growth factor (EGF) receptor (EGFR; Ref. 32), and PP2 (Calbiochem), a at 37°C, placed on ice, washed once with PBS, and lysed in 400 l lysis specific inhibitor of src kinase, (33) were added for 30 min before the buffer (150 mM NaCl, 25 mM Tris (pH 7.5), 1 mM EDTA, 2 mM sodium addition of stimulus. All blocking Abs were used at 10 ␮g/ml. vanadate, 10 mM NaF, 2 mM sodium pyrophosphate, 1% Nonidet P-40, 2 ␮g/ml aprotinine, 2 ␮g/ml leupeptin, 2 mM PMSF, 10% glycerol). Fol- lowing centrifugation for 10 min at 10,000 ϫ g, the protein content in the FACS analysis supernatants was determined using the bicinchoninic acid reagents (Pierce, Rockford, IL). Ab (rabbit polyclonal anti-EGFR Ab, 5 ␮g/sample; Upstate For FACS analysis, HMECs or HUVECs were trypsinized shortly, placed Biotechnology, Lake Placid, NY) was added to each sample containing 0.8 in PBS containing 2% FCS, incubated for 30 min on ice with anti-C5aR or mg protein, and the samples were rotated for2hat4°C. Protein A/G anti-C3aR Ab (1/200 dilution; both from BD PharMingen, San Diego, CA), Sepharose (30 ␮l of a 50% slurry; Amersham Pharmacia Biotech) was washed three times with PBS/FCS, and labeled with mouse- or rabbit- added for 30 min, and the samples were washed three times in lysis buffer biotinylated IgG for 30 min. Following another three washes, the cells were and once in PBS. The resulting pellets were suspended in 30 ␮l SDS incubated with streptavidin-PE (1/250 dilution; BD PharMingen) for 30 sample buffer, boiled for 3 min, and loaded onto a 7% SDS gel. Western min, washed once more, and analyzed on a FACScan using CellQuest Pro transfers and blots were performed according to standard protocols, using software (BD Biosciences, Mountain View, CA). The specificAbwas 5% nonfat dry milk to block the blot, followed by a 1/2000 dilution of the replaced with anti-rabbit (C5aR) or anti-mouse (C3aR) IgG for negative anti-pTyrosin Ab (PY20; BD Transduction Laboratory, Mountain View, controls. CA) and goat anti-mouse HRP-IgG conjugate (BioSource International,

Camarillo, CA) as the second Ab (1/8000 dilution) followed by detection Downloaded from RT-PCR of C3aR and C5aR by ECL (ECL plus reagent; Amersham Pharmacia Biotech). The blots were stripped with Re-Probe (Geno Technology, St. Louis, MO), and redevel- RNA was purified from confluent HUVEC or HMEC cells with the oped with the anti-EGFR Ab. The densitometric ratio between the two RNeasy kit (Qiagen, Valencia, CA) and poly(dT)-tailed cDNA was pro- blots was determined and values were compared with those in unstimulated duced using the Omniscript RT kit (Qiagen). The primers to detect the cells (n ϭ 4). C3aR were derived from the sequences between aa 161 and 340. Forward primer: 5Ј-CGC GAA ATC TTC ACT ACA GAC AAC C-3Ј. Reverse primer: 5Ј-TCA CCT AGT GAT CGT TAT TGC CAC GA-3Ј. The prim- Determination of cell barrier function http://www.jimmunol.org/ ers to detect the C5aR were derived from aa 1–132. Forward primer: 5Ј- AAC ATG AAC TCC TTC AAT TAT ACC ACC CCT G-3Ј. Reverse HMECs (2 ϫ 104 cells/filter) were seeded on 0.4 ␮m pore-size Transwell primer: 5Ј-GGC GCT GAT GGT GGC CAC GAG CAG GAT GCT-3Ј. filters (Costar) and grown to confluence for 7 days. FITC dextran (m.w. The PCR was conducted for 40 cycles using a PerkinElmer Cetus instru- 70,000; Sigma-Aldrich, St. Louis, MO) was added to the upper wells along ment (PerkinElmer, Wellesley, MA). Melting was for 40 s at 94°C, an- with no stimulus, 1 ␮M C3a, or 100 nM C5a; and as a function of time, nealing was for 1 min at 56°C, and chain elongation was conducted at 72°C aliquots were withdrawn from the lower chamber and fluorescence mea- for 2 min. sured using a Packard fluorocount plate reader (Packard Instrument, Me- riden, CT). Maximal flux was determined using filters not covered with cells. Fluorescence microscopy by guest on September 26, 2021 Filamentous actin (F-actin) formation was visualized as previously described for HMECs activated with IL-8 (34). In short, HMECs or HUVECs Eosinophil transmigration were seeded at low density on collagen-coated glass coverslips, grown to Eosinophils were prepared from mildly allergic but otherwise healthy hu- confluence, serum starved for 2 or 4 h, and stimulated with the indicated man donors by Percoll centrifugation followed by negative selection of concentration of C3a or C5a for the times stated. In some experiments, CD16 positive cells by using a magnetic separation technique (35, 36). The HMECs were used just before reaching confluence to allow morphometric cells were labeled fluorescently using 1 ␮M calcein-AM (Molecular analysis of individual cells. The cells were fixed with 3% paraformalde- Probes). hyde in PBS, put on ice and permeabilized for 5 min in 0.2% Triton X-100, HMECs were grown as monolayers on 5-␮m pore-size inserts (Costar). incubated with 5 mU/ml of Alexa 488-phalloidin (Molecular Probes, Eu- In some cases, cytochalasin B (5 ␮g/ml) was added to the cells for 5 min, gene, OR) for 30 min, washed three times with PBS, and mounted with followed by extensive washing with DMEM. Subsequently, 200,000 fluo- Antifade (Molecular Probes). Fluorescence microscopy was performed on ϫ rescently labeled eosinophils were added to each upper well and no me- a Leica DM IRBE microscope using a 100 oil immersion objective. diator, C3a (1 ␮M), or C5a (50 nM) were pipetted into the lower wells. Images were obtained with a Hamamatsu digital camera (Hamamatsu Pho- After 4 h, the number of transmigrated cells in the lower well was counted tonics, Hamamatsu City, Japan) and analyzed with Improvision OpenLab in a central 1.1-mm2 area using a Leitz Fluovert FS microscope (Leica 3.0 software (Improvision, Boston, MA), which allows the determination Microsystems, Deerfield, IL). All values are indicated as relative to those of fluorescence intensity and cell area of individual cells. A minimum of 30 seen in the presence of 1 ␮M C3a, which caused an intermediate level of cells in three different experiments was analyzed for each data point. To chemotaxis. determine cell retraction, HMECS or HUVECs were grown to confluence, stimulated and stained as above, and retracted cells were counted in the digital image representing a 1-mm2 area. Results Haptotaxis of endothelial cells on collagen Expression of the C3aR and the C5aR by endothelial cells The assay was performed as described previously for IL-8 (34). The bottom The presence of C3aRs and C5aRs on endothelial cells was deter- side of Transwell filters (8-␮m pore size; Costar, Cambridge, MA) was mined by FACS analysis with receptor-specific Abs. Both HMECs coated for 30 min with 230 ␮l bovine collagen (100 ␮g/ml; Cohesion, and HUVECs expressed receptors for both the C3aR and the C5aR Franklin, MA) in PBS and blocked for 30 min with 1% BSA. Endothelial (Fig. 1A), although apparent receptor numbers were low in all ␮ ␮ basal medium (500 l/well; Clonetics) containing 0.1% BSA, 0.5 g/ml cases. Because the shift was monophasic, it appears that all hydrocortisone, and 50 ␮g/ml gentamicin was pipetted into the bottom well, and 5 ϫ 104 HMECs or 2.5 ϫ 104 HUVECs in the same media were cells expressed a small number of receptors. This was con- added to the inserts. Following the addition of C3a or C5a to the bottom firmed by the uniform response of all cells in the actin poly- wells, the cells were incubated for4hat37°C in a tissue culture incubator merization assay (Fig. 2). and stained for 10 min with 1 ␮M calcein-AM (Molecular Probes). Cells To verify the results obtained by FACS, messages for C3aR in the upper well were carefully removed with a cotton swab and transmigrated cells were counted at ϫ5 magnification on a Leica DM IRBE and C5aR RNA were amplified using RT-PCR. Products for the microscope (Leica, Deerfield, IL) using FITC excitation and emission. Re- C3aRs and the C5aRs were detected in both HUVECs and sults represent the mean Ϯ SEM of four experiments in triplicate. HMECs (Fig. 1B). 2104 EFFECTS OF C3A AND C5A ON ENDOTHELIAL CELLS Downloaded from

FIGURE 1. Expression of C3aRs and C5Rs by HMECs and HUVECs. A, FACS analysis of the expression of the C3aR and the C5aR by human endothelial cells. Samples were prepared as described in Materials and Methods. Top panel, Results obtained for HMECs; bottom panel, Results for HUVECs. B, RT-PCR of C3aR and C5aR message by HMECs and http://www.jimmunol.org/ HUVECs. Lane 1, C3aR in HMECs (expected size: 0.54 kb); lane 2, C5aR in HMECs (expected size: 0.4 kb); lane 3, 1-kb ladder; lane 4, C3aR in HUVECs; and lane 5, C5aR in HUVECs. FIGURE 2. Time course of the cytoskeletal response to C3a and C5a. Subconfluent HMECS grown on collagen were stimulated with 10Ϫ7 M Ϫ8 Cytoskeletal effects of C3a and C5a on endothelial cells C3a, 10 M C5a, or 100 ng/ml LPA at 37°C, fixed with paraformalde- hyde, stained with Alexa 488 phalloidin, and processed as described in To show that the C3aRs and C5aRs on endothelial cells were func- Materials and Methods. A, Unstimulated HMECs; HMECs stimulated with tional, the effect of C3a and C5a on the cytoskeleton of endothelial C5a for 1 min (B), 5 min (C), 20 min (D), 45 min (E), and 60 min (F), or cells was determined. The addition of C3a or C5a caused increased with C3a for 1 min (G), 5 min (H), or 20 min (I). LPA caused a similar by guest on September 26, 2021 F-actin formation in both HMECs (Fig. 2, top left panel) and response shown at 1 min (J), 5 min (K), and 20 min (L) (all ϫ100 objec- HUVECs (Fig. 2, bottom left panel). In unstimulated serum- tive). Changes similar to those induced by C3a and C5a in HMECs were ϫ starved endothelial cells, F-actin staining of low intensity was con- observed in HUVECs ( 60 objective). M, Unstimulated HUVECs; N, HUVECs stimulated with 10Ϫ7 M C3a for 1 min; and O, HUVECs stim- centrated in the cell periphery, where adjacent cells touched each Ϫ ulated with 10 8M C5a for 20 min. Right panels, Quantification of F-actin other. After the addition of C3a, prominent stress fibers appeared response; C3a stimulation caused a transient increase in stress fiber for- within 1 min of activation, which led to an increase in F-actin mation. In HMECS, this response was best quantified by determining the staining and to stretching of the cells on the substratum. In area covered by individual cells (top right panel, mean Ϯ SEM of 30–40 HMECs, this behavior could best be quantified by determining the cells for each data point). The time course of C3a activation in HUVECs surface area covered by individual cells, which more than doubled closely resembled that in seen in HMECs (bottom right panel, f: mean Ϯ at this early time point (Fig. 2, top right panel). The response to SEM of 30 regions; 2000 ␮m2). The response to C5a was delayed and C3a was transient and almost completely reversed by 5 min. In prolonged, and the increase in polymerized actin was characterized by a contrast, the response to C5a was prolonged lasting for almost 1 h combination of stress fiber formation and cell contraction. Quantification of (Fig. 2) and leading to a 3-fold increase in F-actin content (Fig. 2, these results showed increased F-actin content in both HMECs (middle right panel) and HUVECs (bottom right panel). middle right panel). Starting between 5 and 10 min, it was observed that a fraction of the cells retracted, leaving denuded surface areas between adjacent cells. The affected fraction of cells was not large enough to result in a significant decrease of mean cell protracted response to C5a. In the confluent monolayer formed by surface area (Fig. 2), but when retracted cells were counted on a HUVECs, C5a stimulation induced gap formation between adja- confluent layer of HMECs, an increase of retracted cells from 6 Ϯ cent cells (see Fig. 2O), leaving denuded substratum in between 3 cells/mm2 in unstimulated cells to 63 Ϯ 31 cells/mm2 following cells. The number of contracted cells increased from 4 Ϯ 1 cells in stimulation with 10Ϫ8 M C5a for 20 min was observed (see also unstimulated HUVECs, to 30 Ϯ 3 cells after a 20-min exposure to Fig. 4, C and D). No such effect was noted with C3a. 10Ϫ8 M C5a. Because it was problematic to achieve healthy quiescent Cytoskeletal changes were induced by concentrations of C3a or HUVECs by prolonged serum starvation, these cells were grown to C5a within the low (C5a) to high (C3a) nanomolar range as ex- confluence, serum starved shortly (2 h), and stimulated. Because pected from the dissociation constants of the two receptors for cell borders were difficult to assess using confluent cells, the mean their respective ligands (Refs. 37 and 38; Fig. 3). A 21-aa synthetic phalloidin staining intensity was determined for a 2000-␮m2 area peptide derived from the C terminus of human C3a is reported to covered by several cells. The response to C3a and C5a in HUVECs have similar specific activities for smooth muscle contraction and (Fig. 2, MÐO, and bottom right panel) was very similar to that seen induction of vascular permeability as full-length C3a (1). In vitro, with HMECs, including the transient time course for C3a and the this peptide is one to two orders of magnitude less active than C3a. The Journal of Immunology 2105

measured using FITC-dextran passage through filters bearing monolayers of HMECs stimulated with C3a or C5a. In the absence of the anaphylatoxins, endothelial cells created a nearly imperme- able boundary between the two compartments. After stimulation with C5a, an increased amount of the labeled dextran was able to pass through the cell barrier (Fig. 4E). In contrast, C3a did not perturb the cell barrier function (Fig. 4E). Participation of endothelial cell anaphylatoxin receptors in FIGURE 3. Dose response of the cytoskeletal response of HMECs stim- eosinophil transmigration ulated with C3a (1-min time point) or C5a (20-min time point). These time Because eosinophils can transmigrate through an endothelial cell points were chosen because they represented the maximal response to each monolayer in response to C3a and C5a (36), and because the pres- stimulus. The response to C3a is represented as change in cell area, ence of endothelial cells augments the anaphylatoxin-mediated whereas the response to C5a is quantified as change in fluorescence inten- chemotaxis of the eosinophils, it was important to determine sity. Left panel, Dose response to C3a (f) and the 21-mer C3a (55–75) peptide (Œ) at 1 min; right panel, Dose response to C5a at 20 min. whether endothelial cell activation contributed to this process. To prevent the cytoskeletal response of the endothelial cells, cytochalasin B was added to either naked filters or filters with monolayers In accordance with these reports, the C3a-peptide (57–77) caused of endothelial cells as described previously for epithelial cell trans- Downloaded from the same response as C3a in HMECs, but at ϳ30-fold higher con- migration (39). Following thorough washing, eosinophils were centrations (Fig. 3). added to the upper chambers and anaphylatoxins to the lower chambers, and the assemblies incubated for4hat37°C. Untreated Effect of C3a and C5a on cell retraction and monolayer monolayers of endothelial cells were used for comparison. As re- permeability ported previously for HUVECs (36), the presence of HMECs increased the chemotaxis of eosinophils toward C3a and C5a com- As noted above, C5a, but not C3a, caused retraction of the two pared with naked filters (Fig. 5). To assure that any residual traces http://www.jimmunol.org/ endothelial cell types used (Fig. 4, AÐE). Because stimulation with of cytochalasin B that were left after thorough washing were in- C5a caused cell retraction and gap formation between cells, mono- sufficient to perturb the migratory machinery of the eosinophils, layer permeability was determined next. Paracellular flux was transmigration of eosinophils through cytochalasin B-treated filters was determined and did not result in a negative effect on naked filters (Fig. 5). In contrast, cytochalasin B treatment of the HMEC monolayers reduced eosinophil chemotaxis induced by C5a to levels seen in the absence of endothelial cells (Fig. 5), indicating that the cytoskeletal response of the endothelial cells contributed to the C5a-mediated transmigration of eosinophils. The contribution of by guest on September 26, 2021 the endothelial cell cytoskeletal response to C3a-dependent chemotaxis was only minor. Signal transduction cascade used by the C3aRs and C5aRs The relative ease of the actin staining assay and the availability of specific inhibitors for components of the signal transduction machinery allowed to screen for downstream elements of the response

FIGURE 4. Effect of C3a and C5a on endothelial cell retraction and paracellular flux. AÐE, Effect of anaphylatoxins on cell retraction. Conflu- FIGURE 5. Influence of the endothelial cell cytoskeleton on eosinophil ent monolayers of HMECs or HUVECs were stimulated with 100 nM C3a transmigration. Confluent monolayers of HMECs grown on 5-␮m pore- or 10 nM C5a and stained for F-actin as described in Materials and Meth- size inserts were incubated with 5 ␮M cytochalasin B or 0.01% DMSO ods. Retracted cells were counted using a ϫ10 objective. A, HMECs, un- vehicle for 5 min and thoroughly washed. Empty filters were treated the stimulated; B, HMECs stimulated with C3a for 20 min; C, HMECs stim- same way. C3a (1 ␮M) or C5a (50 nM) was added to the bottom wells and ulated with C5a for 20 min; D, Quantitation of retracted HMECs (mean Ϯ 200,000 fluorescently labeled eosinophils were added to the top compart- SD for 12–16 square areas each). E, Quantitation of retracted HUVECs ments. After4hat37°C, chemotaxis of eosinophils was determined as (mean Ϯ SD for 12 square areas). Ⅺ, No stimulus; z, ϩC3a; f, ϩC5a. F, desribed in Materials and Methods. Ⅺ, Empty filters, mock treated; f, Effect of C3a and C5a on paracellular flux. Leakage of 70,000 m.w. FITC- empty filters treated with cytochalasin B; z, mock-treated HMEC mono- dextran through confluent monolayers of HMECS grown on 0.4-␮m pore- layers; p, cytochalasin B-treated HMECs (mean Ϯ SD of four experi- size filters was determined as described in Materials and Methods. E, ments). Although cytochalasin B treatment of empty filters had no negative Unstimulated HMECs; , HMECs stimulated with C3a; ƒ, HMECs stim- effect on eosinophil transmigration, it blocked transmigration through en- ulated with C5a. Maximal dye flux was determined on filters not coated dothelial cell monolayers to the levels observed in the absence of endo- with cells (F). One experiment representative of four. thelial cells. 2106 EFFECTS OF C3A AND C5A ON ENDOTHELIAL CELLS to C3a and C5a. In leukocytes, both C3aRs and C5aRs couple to

Gi␣, which is abundant in hemopoietic cells. Endothelial cell C5aRs similarly coupled to Gi␣, as indicated by the blocking effect of PT on the cytoskeletal response to C5a in both HMECs and HUVECs (Fig. 6, C and H). In contrast, the effect of C3a was if anything accentuated following preincubation with PT (Fig. 7, F and X), implying use of a different G protein for the C3aR in endothelial cells. Because stress ﬁber formation has been associated with activation of rho (40), the effect of C3 botulinum toxin, which blocks rho (30), and Y27632, which inhibits rho kinase (31), were determined next. Both of these inhibitors blocked the C3a-mediated cytoskeletal response in HMECs (Fig. 7, B and D), but failed to prevent the C5a-dependent effect (Fig. 6B), indicating that only the C3aR activates the rho cascade. The same was seen in HUVECs, where Y27632 abrogated the response to C3a (Fig. 6W). Similarly, C3 botulinum toxin blocked this response (data not shown). However, it was noticed that in the presence of C3 toxin, the integrity of the HUVEC cell monolayer was disturbed. Although we used condi- Downloaded from tions that were the same as those used previously to block rho in HUVECs (30, 41), the C3 toxin results in these cells should be seen with caution. Recent reports indicate that there is considerable cross talk between GPCRs and receptor tyrosine kinases (42, 43), and that pro- http://www.jimmunol.org/

FIGURE 7. Effect of inhibitors on the C3a-mediated cytoskeletal response in HMECs (AÐJ) and HUVECs (VÐZ) at 1 min. A and B, Inhibitor, 10 ␮M Y27632; A, no stimulus; B, ϩ100 nM C3a; C and D, inhibitor, 10 ␮g/ml C3 botulinum toxin for 24 h (30); C, no stimulus; D, ϩ100 nM C3a; E and F, inhibitor, 100 ng/ml PT for 16 h; E, no stimulus; F, ϩ100 nM by guest on September 26, 2021 C3a; G and H, inhibitor, 1 ␮M AG1478; G, no stimulus; H, ϩ100 nM C3a; I and J, inhibitor, 1 ␮M PP2; I, no stimulus; J, ϩ100 nM C3a. In the middle graph, the effect of the various inhibitors is quantiﬁed (mean Ϯ SEM of 30–40 cells each) using cell area determination as the parameter measured. C3 botulinum toxin and Y27632 showed statistically signiﬁcant inhibition (p Ͻ 0.01) of the cytoskeletal response to C3a. For comparison, LPA (100 z ng/ml) known to couple to G␣13 (61) was used in the same assay ( ). VÐZ, HUVECs stimulated with 100 nM C3a for 1 min. V, No inhibitor; preincubation with 10 ␮M Y27632 for 30 min (W), with 100 ng/ml PT for 16 h (X), with 1 ␮M AG1478 (Y), or 1 ␮M PP2 (Z) for 30 min. Bottom panel, The mean Ϯ SEM of 30 2000-␮m2 rectangles each is shown. There is a statistical difference for Y27632-treated cells (p Ͻ 0.01).

tracted responses of GPCRs often involve the secondary activation of receptor tyrosine kinases such as the EGFR. Therefore, AG1478, a specific inhibitor of the EGFR tyrosine kinase activity (44), and PP2, a specific inhibitor of src kinase (33), were tested in FIGURE 6. Effect of inhibitors on the cytoskeletal response to C5a: the actin polymerization assay. In the two endothelial cell types HMECs (AÐE) or HUVECs (FÐJ) were incubated with inhibitors as de- tested, both inhibitors blocked the actin response to C5a (Fig. 6, D, scribed for Fig. 6, and then stimulated with 10 nM C5a for 20 min. A and E, I, and J), but not to C3a (Fig. 6, H, J, Y, and Z). All inhibitors F, ϩC5a, no inhibitor; B and G, ϩC5a ϩ Y27632; C and H, ϩC5a ϩ PT; of the C5a actin response also prevented cell retraction (Fig. 6, D and I, ϩC5a ϩ AG 1478; E and J, ϩC5a ϩ PP2. Middle and bottom right middle and bottom panels). panels, The results were quantified for HMECs and HUVECs, respectively. Left panels, The mean F-actin fluorescence intensity (mean Ϯ SEM, n ϭ Effect of C3a and C5a on endothelial cell migration 30–40). PT, AG 1478, and PP2 showed a statistically significant inhibition Because actin polymerization is a prerequisite for cell migration, (p Ͻ 0.01) in F-actin content in both cell types, while Y27632 failed to prevent the C5a-mediated response, although it led to finger-like projec- we questioned whether HMECs and HUVECs were chemoat- tions not seen in control cells. Right panels, The effect of the same inhib- tracted by C3a or C5a. Although C3a failed to cause endothelial itors on cell retraction, indicating the same inhibition profile (mean Ϯ SD, cell migration, C5a showed a chemotactic effect on the endothelial n ϭ 12–16). PT slightly enhanced the number of retracted cells in the cells (Fig. 8A). Cell migration was blocked by the same inhibitors absence of C5a, but prevented the C5a-induced increase in cell retraction. that prevented the C5a-mediated actin response, i.e., PT (Fig. 8, C The Journal of Immunology 2107 Downloaded from http://www.jimmunol.org/

FIGURE 8. C5a-mediated cell migration of endothelial cells involves activation of the EGFR. A and B, C5a, but not C3a, is chemotactic for endothelial cells. To determine cell migration, calcein-labeled HMECs (50,000 cells/well) or HUVECs (25,000 cells/well) were added to the upper compartment of collagen-coated 8-␮m pore-size Transwell filters. Following the addition of varying concentrations of C3a or C5a to the lower compartments, cells were incubated for4hat37°C. Cells that adhered to the underside of the filters were counted under a fluorescent microscope. Ⅺ, Cell migration in the presence of C5a; f, cell migration in the presence of C3a. Mean Ϯ SD of 6–12 filters. C and D, Effect of various inhibitors of the EGFR cascade on C5a-mediated migration of HMECs and HUVECs. Cell migration was determined as described above. Inhibitors were added for 30 min before the addition of 30 nM C5a or 50 ng/ml EGF as a positive control. Ⅺ, No inhibitor; f,1␮M AG 1478; z, neutralizing anti-EGF Ab (MAB236, 10 ␮g/ml; R&D Systems,

Minneapolis, MN); `, anti-HB-EGF Ab (rabbit polyclonal, 10 ␮g/ml; R&D Systems); o, neutralizing anti-EGFR Ab (LA1, 10 ␮g/ml; Upstate Biotech- by guest on September 26, 2021 nology); ␯,1␮M PP2. In addition, the effect of PT is also shown (s). Mean Ϯ SD of 6–12 filters. E and F, Phosphorylation of the EGFR following stimulation with C5a. HMECs were stimulated with 100 nM C5a or 50 ng/ml EGF for the indicated times and immune precipitated with anti-EGFR Ab (rabbit polyclonal; Upstate Biotechnology) as described in Materials and Methods. Proteins in the precipitates were separated on SDS-PAGE, Western transferred and immunoblotted with anti-phospho-Tyr Ab, then reprobed with anti-EGFR Ab to assure equivalent protein loading. Left panel, Western blot; right panel, Quantification of EGFR phosphorylation adjusted for receptor loading (mean Ϯ SEM, n ϭ 4). and D), PP2, and AG1478 (Fig. 8, C and D), indicating that C5a- lium during the inflammatory process has not been sufficiently mediated cell migration of endothelial cells—in contrast to C5a- appreciated. A number of receptors involved in leukocyte chemo- mediated chemotaxis of leukocytes—depended on EGFR transac- taxis, e.g., the CXCR1, CXCR2, CXCR4, and CCR1, are also tivation. The effect of PP2 appeared to be distal of the EGFR expressed by endothelial cells, especially by microvascular endo- activation, as this inhibitor also blocked EGF-mediated cell mi- thelial cells (34, 46–48). C3a and C5a add another pair of GPCRs gration. To exclude any possible nonspecific effects of AG1478, a expressed both by the endothelium and by leukocytes, and the neutralizing Ab against the extracellular domain of the EGFR was relative role of receptors on both cell types deserves further inves- used as a second way of inhibiting EGFR function. This Ab tigation. Interestingly, it has been described that only C5a, but not blocked C5a-dependent endothelial cell migration (Fig. 8B), which C3a, induced eosinophil transmigration in vivo in the rabbit mes- also indicated that inside-out signaling to the EGFR was involved. enteric artery model (36), although both C3a and C5a were che- Finally, blocking Abs against heparin-binding EGF-like growth moattractant for these cells in vitro. It is possible that endothelial factor (HB-EGF), but not against EGF, inhibited C5a-dependent cell retraction caused by the stimulation of endothelial cells with endothelial cell migration (Fig. 8, C and D), indicating that the C5a played a role in this process, while stimulation with C3a did transactivation of the EGFR was mediated by generation of HB- not allow leukocyte penetration. Both C3a and C5a are known to EGF, as previously described for the thrombin receptor (45). To increase vascular permeability, and although this may in part be an prove transactivation of the EGFR, C5a-stimulated HMECs were indirect effect following mast cell degranulation (7, 8, 49), our data immunoprecipitated with anti-EGFR Ab and phosphorylation of suggest that endothelial cell retraction may also contribute to this the receptor could be detected by Western blotting with an anti- phenomenon. phosphotyrosine Ab (Fig. 8, E and F). Generally, factors which cause increases in endothelial cell monolayer permeability and cell migration are also angiogenic Discussion (50). This has been shown for vascular endothelial cell growth This study shows that endothelial cells express functional C3aRs factor (51), thrombin (52), and IL-8 (53, 34). Our results suggest and C5aRs and delineates the different signal transduction path- that it is possible that C5a may have angiogenic properties, which ways induced by the two stimuli. This active role of the endothe- have not been recognized. However, a putative angiogenic effect of 2108 EFFECTS OF C3A AND C5A ON ENDOTHELIAL CELLS

C5a could only operate in a situation of chronic inflammation with cursor that becomes activated by proteolytic cleavage during cell sustained complement activation, because the activity of C5 con- activation (70) and goes on to activate the EGFRs HER1 and vertase is short-lived (36, 54), and because C5a is rapidly con- HER4. Expression of HB-EGF is up-regulated in cells undergoing verted by plasma carboxypeptidase N to C5a desArg (55), which stress or exposed to proinflammatory stimuli (71–73). Although it has a lower affinity for the C5aR. is not clear whether the concentration of HB-EGF is the rate-lim- In this study, the cytoskeletal response to anaphylatoxins was iting factor during GPCR-mediated transactivation of the EGFR, it studied extensively in large vessel endothelial cells (HUVECs) and is appealing to speculate that its up-regulation may amplify the a dermal microvascular endothelial cell line (HMECs), but it was response to various GPCRs during the inflammatory response. not limited to these cells. It could in fact be seen in all endothelial Complement activation leads to the concomitant generation of cell types tested, which included human brain microvascular cells C3a and C5a in vivo, and concentrations of C3a exceed concen- (a gift from Dr. M. Fiala, University of California, Los Angeles, trations of C5a by about an order of magnitude. Because endothe- CA) and pulmonary microvascular endothelial cells (HMVECs; lial cell activation was similarly an order of magnitude more sen- Clonetics; data not shown). sitive to C5a than C3a, the C3aR and C5aR pathways would be

In leukocytes, both the C3aRs and C5Rs couple to G␣i and G␣16 activated concomitantly. Similar dual endothelial cell activation (56, 57, 21, 26). G␣16 expression is limited to hemopoietic cells, pathways exist for IL-8 and for thrombin, although they are reg- and G␣i is unusually abundant in these cells. Because G protein ulated at different levels. Instead of activation by two ligands, C3a coupling appears to be far less specific than initially recognized, it and C5a, IL-8 activates two receptors, the CXCR1, which behaves is not surprising that G protein usage appears to vary for different like the C3aR and activates rho, and the CXCR2, which behaves cell types. Specifically, activation of the C3aR on endothelial cells like the C5aR (34) and stimulates the EGFR cascade (I. U. Downloaded from was not inhibited by PT, implying that the receptor couples to a Schraufstatter, K. Trieu, D. Rose, R. A. Terkeltaub, and M. different G protein in these cells. Because inhibitors of the rho Burger, manuscript in preparation). Thrombin achieves the same cascade blocked C3a function, these G proteins are likely G␣12 responses (74) by concomitant coupling to several G proteins, in- and/or G␣13, which are both known to activate rho (58). Interest- cluding Gq,G␣i, and G␣12 (75, 76, 45). ingly, a recent report indicated that activation of the thrombin re- An active role of the endothelium in leukocyte transmigration ceptor in the same endothelial cell line used in this study could that goes beyond the presentation of adhesion molecules has only http://www.jimmunol.org/ cause coupling of that receptor to G␣i,G␣q,G␣12, and G␣13. How- been recognized recently (77, 78). As in the current study, these ever, only competitive inhibition of G␣12 or G␣13 could prevent reports found that the cytoskeletal response of endothelial cells stress fiber formation (59). Furthermore, the response to C3a was assisted in the passage of leukocytes (77–79). In the case of lym- short-lived, as has been described previously for lysophosphatidic phocyte transmigration through brain endothelial cell monolayers, acid (LPA) and endothelin, two mediators that couple to G␣13 and rho activation was essential (78), which was not the case in our G␣12, respectively, and signal through rho stimulation (60, 61). experiments. Future experiments will have to show whether this Importantly, activation of HMECs with C3a closely resembled the difference was determined by the type of leukocyte, the type of response to LPA (Figs. 2 and 6). Coupling of the C3aR to G␣12 or endothelial cell, or a difference in the duration of the activation, by guest on September 26, 2021 G␣13 rather than to G␣i would also explain the lack of a chemo- which was more transient in our case. tactic response of endothelial cells stimulated by C3a, because In summary, C3aRs and C5aRs on endothelial cells may con- chemotaxis appears to be a consequence of G␣i activation (62). tribute in various ways to the up-regulation of inflammatory pro- Activation of endothelial cell C5aRs in contrast led to coupling cesses observed following complement activation. to G␣i as supported by the inhibitory effect of PT and the migratory response induced by C5a. Interestingly, a large portion of the en- Acknowledgments dothelial cell response to C5a was blocked by various modes of We thank Dr. Tony Hugli for the C3a peptide and for helpful discussions inhibition of the EGFR. In leukocytes, which lack EGFRs, the and Dr. Milan Fiala for the brain microvascular endothelial cells. signal transduction machinery of the C5aR is sufficient to cause cell migration directly. It is not clear at this point why there is this References difference in the cell activation response of these different cell 1. Hugli, T. E. 1984. Structure and function of the anaphylatoxins. Springer Semin. Immunopathol. 7:193. types. Transactivation of the EGFR by activation of GPCRs has 2. Hugli, T. E. 1990. Structure and function of C3a anaphylatoxins. Curr. Top. been described over the last few years for a number of GPCRs (63, Microbiol. Immunol. 153:181. 3. Hugli, T. E. 1986. Biochemistry and biology of anaphylatoxins. Complement 42, 45), but it is certainly not a ubiquitous finding for all GPCRs. 3:111. It has not been described previously for the C5aR, but may have 4. Fernandez, H. N., P. M. Henson, A. Otani, and T. E. Hugli. 1978. Chemotactic long-reaching consequences in vivo. 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